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Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVI | Pages 21 - 21
1 Aug 2012
Khan A Surrao D Waldman S
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Bioreactors used in tissue engineering are mostly batch-fed with media added and removed periodically. Continuous flow bioreactors help increase ECM accumulation and cell proliferation, due to continuous flow of fresh media, thus, maintaining a steady extracellular nutrient environment. In previous work, we found chondrocytes cultured in continuous flow bioreactors with 20mM HEPES, accumulated considerably more matrix than static cultures. Hence, the objective of this study is to determine if NaHCO3 helps maintain a more physiological extracellular pH in the bioreactor, thus, enhancing ECM accumulation.

Cartilaginous tissue constructs were generated from isolated chondrocytes harvested from the metacarpal joints of 12-18 month old calves. Cells were seeded in high-density 3D cultures (2 million cells/construct). Constructs were cultivated in a continuous flow bioreactor, with and without 14 mM NaHCO3 supplemented media, for 5 weeks, at 37 degrees Celsius, 95% relative humidity and 5% CO2. After 5 weeks of culture the tissue weight, thickness, pH and ECM deposition were determined.

From the results obtained (Table 1), it is evident that chondrocytes cultured in the continuous flow bioreactor with 14mM NaHCO3 and 20mM HEPES, proliferated more extensively and produced more ECM than chondrocytes cultured in only 20mM HEPES. Additionally, the NaHCO3 constructs accumulated ECM in both the vertical (thickness) and horizontal (outgrowth) planes. The question then arises, are the effects mediated by improved buffering, or by addition of NaHCO3 itself. There was a significant difference between the pH of media with (pH 7.41) and without NaHCO3 (pH 6.95) supplementation, with no exposure to cells or tissue; when allowed to equilibrate with 5% CO2 at 37 degrees Celsius. However, there was little difference between the media after exposure to cells; after five weeks of culture in the bioreactor (Table 1). Thus, in the bioreactor with bicarbonate present, because of increased cell number and activity, the pH fell 0.54 pH units during the 7 hour residence time in comparison to the bioreactor with no bicarbonate supplementation. With no NaHCO3 supplementation, the extracellular pH of the medium fed to the cells was never above pH 7.0 (Table 1); low pH could account, at least in part, for lower ECM and cell numbers.